Veto cells effective in preventing graft rejection and devoid of graft versus host potential

ABSTRACT

A method of transplanting a transplant derived from a donor into a recipient is disclosed. The method comprises the steps of (a) transplanting the transplant into the recipient; and (b) administering to the recipient a dose including non-alloreactive anti-third party cytotoxic T-lymphocytes (CTLs), wherein the non-alloreactive anti-third party CTLs are generated by directing T-lymphocytes of the donor against a third party antigen or antigens, the dose is substantially depleted of T-lymphocytes capable of developing into alloreactive CTLs, thereby preventing or ameliorating both graft rejection by the recipient and graft versus host disease.

FIELD AND BACKGROUND OF THE INVENTION

[0001] The present invention relates to veto cell preparations, methodsof their manufacture and transplantation method using same which can beused to prevent or ameliorate immune rejection of donor organs, tissuesor cells without inducing graft versus host disease (GVHD). Moreparticularly, the present invention relates to a cell preparation foruse in transplantation which preparation includes cells which arefunctional as veto cells but which are substantially devoid ofalloreactive cells.

[0002] Transplantation of allogeneic and xenogeneic organs, tissues andcells is commonly practiced in humans in order to alleviate numerousdisorders and diseases.

[0003] For example, bone marrow (BM) transplantation is increasinglyused to treat a series of severe diseases in humans, such as forexample, leukemia. However, bone marrow transplantation is limited bythe availability of suitable donors, since transplanted tissues musttraverse major histocompatibility barriers which can otherwise lead tograft rejection.

[0004] In view of such limitations, several approaches for enhancinggraft acceptance have been suggested.

[0005] In one approach, cancer patients receiving autologous BMtransplantation were treated with granulocyte colony-stimulating factor(G-CSF), resulting in mobilization of pluripotential stem cells from themarrow to the blood thereby increasing the number of cells which can becollected for autologous transplantation.

[0006] In another approach, major histocompatibility barriers in BMtransplantation in leukemia patients were overcome by using a very largedose of stem cells, preferably a dose at least 3-fold greater thanconventional doses used in T-cell-depleted BM transplantation, inparticular a megadose of CD34+ hematopoietic progenitors (U.S. Pat. No.5,806,529 to Reisner et al.).

[0007] Although the megadose approach facilitated permanent acceptanceof allogeneic donor type skin grafts in mice¹, such an approach is notreadily applicable for human transplantation since the number of stemcells which are required to attain this desirable goal may not be easilycollected from human donors.

[0008] A difficult barrier for the engraftment of donor hematopoieticcell transplantation arises from the marked level of host hematopoieticand immune cells surviving mild preparatory regimens. Several studieshave shown that this challenge can be successfully addressed in rodentsby using large doses of bone marrow cells, adequately depleted ofT-cells and utilized in conjunction with one form or another oftolerance inducing cells, also termed as veto cells.

[0009] Veto cell activity is defined as the capacity to specificallysuppress cytotoxic T-cell precursors (CTL-p) directed against antigensof the veto cells themselves, but not against third party antigens²Several veto cells or bone marrow transplantation facilitating cellscapable of suppressing cytotoxic T-cell precursors have beendescribed³⁻¹¹

[0010] Interestingly, it has been shown that some of the most potentveto cells are of T-cell origin, and in particular a very strong vetoactivity was documented for CD8+ CTL lines or clones¹²⁻¹⁶.

[0011] The specificity of CTL veto cells was demonstrated by severalstudies to be unrelated to their T-cell receptor specificity¹⁷⁻¹⁹.

[0012] The suppression of effector CTL-p directed against the veto cellsis both antigen-specific and MHC-restricted. This suppression resultsfrom the unidirectional recognition of the veto cell by the respondingcytotoxic T-lymphocytes¹⁸. Furthermore, it has been shown that thissuppression is mediated by apoptosis^(18, 20).

[0013] Blocking experiments conducted with anti-CD8 or anti class Iantibodies indicated that the elimination of host anti-donor CTL-p isinduced via an interaction of CD8 molecules on the CTL veto cells withthe a3 domain of class I molecules on the host CTL-p's¹⁸. Support tothis observation was provided by studies in which CD8 eDNA wasintroduced into clones lacking CD8¹⁸. Further support was provided byexperiments which demonstrated that CD8 molecules on the veto cells candirectly induce apoptosis in effector cells²¹. More recently, Asiedu etal. demonstrated that antibody mediated cross linking of CD8 on primatebone marrow veto cells, leads to an increased TGFβ production whichinduces apoptosis in the effector cells²². Alternatively, it has beensuggested that mouse bone marrow veto cells can induce apoptosis viaFas-Fas-L interaction²³.

[0014] The studies described hereinabove demonstrated that veto T-cellpreparations can greatly facilitate graft tolerance in bone marrowtransplantation. However, the veto T-cell preparations are inadequatefor generating graft tolerance since such preparations still include asubstantial amount of alloreacting donor T-cells which can lead to GVHD,thus limiting the successful implementation of this approach.

[0015] Reisner et al., ²⁴, ²⁸ describe the preparation ofnon-alloreactive anti-third party CTLs which can be used to enhancegraft acceptance in mice. However, following the publication of thisabstract and a more careful study, it was realized that the CTLpreparation described therein is not depleted of T-cells capable ofdeveloping post transplantation into anti-host CTLs inflicting GVHD.Thus, this approach per se is not applicable for application in humans.

[0016] There is thus a widely recognized need for, and it would behighly advantageous to have, a novel veto cell preparation devoid ofalloreactivity which can be used for substantially reducing rejection oftransplanted organs, tissues or cells without generating GVHD, therebyleading to durable tolerance towards the transplanted, organs tissues orcells.

SUMMARY OF THE INVENTION

[0017] According to one aspect of the present invention there isprovided a method of transplanting a transplant derived from a donorinto a recipient, the method comprising the steps of (a) transplantingthe transplant into the recipient; and (b) administering to therecipient a dose including non-alloreactive anti-third party cytotoxicT-lymphocytes (CTLs), wherein the non-alloreactive anti-third party CTLsare generated by directing T-lymphocytes of the donor against a thirdparty antigen or antigens, the dose is substantially depleted ofT-lymphocytes capable of developing into alloreactive CTLs, therebypreventing or ameliorating both graft rejection by the recipient andgraft versus host disease.

[0018] According to another aspect of the present invention there isprovided a method of treating a recipient suffering from a diseaserequiring immature hematopoietic cell transplantation, the methodcomprising the steps of (a) conditioning the recipient under sublethal,lethal or supralethal conditions; (b) administering to the recipient afirst dose including immature hematopoietic cells including stem cellsfrom an allogeneic or xenogeneic donor; and (c) administering to therecipient a second dose including non-alloreactive anti-third partycytotoxic T-lymphocytes (CTLs), wherein the CTLs are generated bydirecting T-lymphocytes derived from the donor against a third partyantigen or antigens, the second dose is substantially depleted ofT-lymphocytes capable of developing into alloreactive CTLs, therebypreventing or ameliorating both graft rejection and graft versus hostdisease.

[0019] According to yet another aspect of the present invention there isprovided a method of producing non-alloreactive anti-third partycytotoxic T-lymphocytes (CTLs), the method comprising the step ofdirecting T-lymphocytes against a third party antigen or antigens, andsubstantially depleting T-lymphocytes capable of developing intoalloreactive CTLs.

[0020] According to still another aspect of the present invention thereis provided a cell preparation for transplantation to a recipient, thecell preparation comprising donor derived non-alloreactive anti-thirdparty cytotoxic T-lymphocytes (CTLs) directed against a third partyantigen or antigens, the cell preparation being substantially depletedof T-lymphocytes capable of developing into alloreactive CTLs.

[0021] According to an additional aspect of the present invention thereis provided a cell preparation for transplantation to a recipient, thecell preparation comprising (a) donor derived immature hematopoieticcells including stem cells; and (b) donor derived, non-alloreactiveanti-third party cytotoxic T-lymphocytes (CTLs) directed against a thirdparty antigen or antigens, the cell preparation being substantiallydepleted of T-lymphocytes capable of developing into alloreactive CTLs.

[0022] According to further features in preferred embodiments of theinvention described below, the dose, T-lymphocytes or preparation issubstantially depleted of CD4 T cells and/or CD56 natural killer cells.

[0023] According to still further features in the described preferredembodiments depletion of T-lymphocytes capable of developing intoalloreactive CTLs is effected by deprivation of a factor which is (i)required for CTLs maturation; and (ii) secreted by maturing CTLs.

[0024] According to still further features in the described preferredembodiments the factor is a cytokine.

[0025] According to still further features in the described preferredembodiments the cytokine is IL2.

[0026] According to still further features in the described preferredembodiments depletion of T-lymphocytes capable of developing intoalloreactive CTLs is effected by affinity labeling followed by labelbased separation.

[0027] According to still further features in the described preferredembodiments depletion of T-lymphocytes capable of developing intoalloreactive CTLs is effected by affinity purification.

[0028] According to still further features in the described preferredembodiments the donor is selected from the group consisting of anallogeneic donor either HLA identical or HLA non-identical and axenogeneic donor.

[0029] According to still further features in the described preferredembodiments the recipient is a human.

[0030] According to still further features m the described preferredembodiments the recipient and the donor are both humans.

[0031] According to still further features in the described preferredembodiments the third party antigen or antigens is selected from thegroup consisting of third party cells, a cell antigen, a viral antigen,a bacterial antigen, a protein extract and a purified protein. In apresently preferred embodiment the third party antigen are syntheticpeptides which constitute viral antigens and which are presented byautologous antigen presenting cells pulsed therewith.

[0032] According to still further features in the described preferredembodiments the viral antigen is an EBV or a CMV antigen.

[0033] According to still further features in the described preferredembodiments the purified protein is ovalbumin.

[0034] According to still further features in the described preferredembodiments the third party cells are allogeneic or xenogeneic cellswith respect to the recipient.

[0035] According to still further features in the described preferredembodiments the allogeneic cells have HLA antigens different from thatof the donor but which are not cross reactive with the recipient HLAantigens.

[0036] According to still further features in the described preferredembodiments the allogeneic cells are stimulatory cells selected from thegroup consisting of cells purified from peripheral blood lymphocytes(PBLs), spleen or lymph nodes, cytokine-mobilized PBLs and in vitroexpanded antigen-presenting dendritic cells (APC).

[0037] According to still further features in the described preferredembodiments the immature hematopoietic cells including stem cells arederived from the bone marrow, mobilized peripheral blood, fetal liver,yolk sac and/or cord blood of the donor.

[0038] According to still further features in the described preferredembodiments the mobilized peripheral blood cells are obtained byleukapheresis of peripheral blood of the donor after stimulation with asuitable cytokine.

[0039] According to still further features in the described preferredembodiments the immature hematopoietic cells are T-cell depletedhematopoietic progenitor cells.

[0040] According to still further features in the described preferredembodiments the T-cell depleted hematopoietic progenitor cells are CD34+progenitor hematopoietic cells.

[0041] According to still further features in the described preferredembodiments a cell ratio between the cytotoxic T-lymphocytes and theimmature hematopoietic cells including stem cells is at least 1 to 100,preferably 1.5 to 100.

[0042] According to still further features in the described preferredembodiments the cell number of the cytotoxic T-lymphocytes infused to arecipient is more than 5×10⁶/Kg body weight and the immaturehematopoietic cells including stem cells (CD34 cells) is more than1.0×10⁶/Kg body weight.

[0043] According to still further features in the described preferredembodiments steps of transplanting and administering are effected at thesame time, or alternatively, the step of transplanting is performedeither prior to, or after the step of administering.

[0044] The present invention successfully addresses the shortcomings ofthe presently known configurations by providing veto cells which arehighly effective in preventing graft rejection, yet are devoid of graftversus host disease potential.

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this re, no attempt is made to show details of theinvention in more detail than is necessary for a fundamentalunderstanding of the invention, the description taken with the drawingsmaking apparent to those skilled in the art how the several forms of theinvention may be embodied in practice.

[0046] In the drawings:

[0047]FIG. 1 is a graph depicting the level of anti-host CTLp in donoranti-third party CTL lines prepared with and without IL2 starvation.

[0048]FIG. 2a is a histogram depicting the veto activity ofnon-alloreactive anti third-party CTLs. The Figure shows activity ofanti-third party CTLs as a function of veto cell concentration ascompiled from 17 different experiments. Specific lysis exhibited byresponder cells (C3H/Hej) was documented upon stimulation against Balb/c(solid squares) or SJL (μopen squares) splenocytes, in the presence ofdifferent concentrations of cells from a Balb/c anti third-party(C57BL/6) CTL line.

[0049]FIG. 2b is a graph depicting the veto effect of non-alloreactiveanti third party CTLs as a function of effective veto/responder cellratios. The killing of responder cells (CTL progenitors) (C3H/Hej)stimulated against Balb/c (solid circle) or SJL (open circle)splenocytes, was tested in the presence of different concentrations ofcells from a Balb/c anti third party (C57BL/6) CTL line.

[0050]FIG. 3 is a graph depicting the veto activity of anti third partyCTLs as a function of the time of veto cell addition. The percentkilling of responder cells (C3H/Hej) stimulated against Balb/csplenocytes by a Balb/c anti third party (C57BL/6) CTL line (At a 1:50vetolresponder cell ratio), was tested as a function of the time of CTLaddition.

[0051]FIG. 4 is a histogram depicting the inhibition of the veto effectby a monoclonal anti-CD8 antibody. Anti-CD8 mAb (anti Ly-2.2) was addedat different concentrations to mixed lymphocyte reaction (MLR) whichconsisted of responder cells (C3H/Hej) stimulated against Balb/csplenocytes, and cells from a Balb/c anti-third party (CS7BL/6) CTL line(At a 1:50 veto/responder cell ratio).

[0052]FIGS. 5a-c depict the specific deletion of responder CTL-pdirected against the H-2 antigen of the veto cells. Splenocytes from 2 ctransgenic mice (H-2^(b)) bearing transgene TCR specific against H-2^(d)(1B2⁺) were stimulated in MLR against Balb/c splenocytes (H-2^(d)). Thefrequency of 1B2⁺ CD8 T cells before (5 a) and after addition to the MLRculture of Balb anti-C3H (5 b) or SJL anti-C3H (Sc) CTLs, was determinedby FACS 5 days after initiation of the MLR.

[0053]FIG. 6a is a histogram depicting the role of Fas ligand (Fas-L)and perforin in the veto activity of anti-third party CTLs. Anti-thirdparty CTLs generated from wild type C3H splenocytes were compared toCTLs generated from Fas-L deficient gld/C3H or from perforin deficientPO splenocytes. Cultures for veto determination consisted of Balbanti-C3H MLR (solid column) while Balb anti-SJL served as a control forbackground inhibition (open column). In the case of PO CTLs, culturesfor veto determination consisted of C3H anti-PO MLR (solid column) whileC3H anti-SJL served as a control (open column).

[0054]FIG. 6b is a histogram depicting the reversal of veto activity inFas-L deficient CTLs by gene transfer of Fas-L. Anti third-party CTLsgenerated from wild type C3H splenocytes, were compared to CTLsgenerated from Fas-L deficient gld/C3H, before, a, and aftertransfection with Fas-L containing retroviral vector, b, or with a mockvector, c.

[0055]FIG. 7a is a histogram depicting the role of Fas in the vetoactivity of anti-third party CTLs. Responder CTL progenitors of wildtype C3H or of Fas deficient lpr/C3H background, were compared for theirsensitivity to the veto effect of Balb anti-C57BL/6 CTLs. Cultures forveto determination consisted of C3H anti-Balb MLR (solid columns) orlpr/C3H anti-SJL (open columns) which served as a control fordetermining background inhibition.

[0056]FIG. 7b is a histogram depicting the effect of a Fas-L antagoniston the veto effect of CTLs. Cultures for veto determination consisted ofBalb anti-C3H MLR (solid columns) or Balb anti-SJL (open column) whichserved as a control for determining background inhibition.

[0057]FIGs. 8a-d depict the deletion of anti-H2^(d) T-cells bynon-alloreactive anti-third party CTLs. Splenocytes from 2 c transgenicmice (H-2^(b)) bearing transgene TCR specific against H-2^(d) werestimulated in MLR against Balb/c splenocytes (H-2^(d)). Expression ofFas on CD8 transgenic (1B2⁺) T cells was analyzed by FACS before MLR(FIG. 8a) and at 48 hr (FIG. 8b). Deletion of Fas⁺ 1B2⁺ CD8 T-cellsfollowing addition to the MLR culture of Balb anti-C3H (FIG. 8c) or SJLanti-C3H (FIG. 8d) CTLs, was determined at 48 hrs after initiation ofthe MLR.

[0058]FIG. 9 is a graph depicting the relationship between cells perculture and the % of non-responding cells of unmanipulated peripheralblood lymphocytes (PBLs) taken from a donor (R) and stimulated againstpotential host type PBL (patient No. 38). The specific killing ofHLA-unrelated cell types (patient No. 40) was demonstrated to benegligible (square), while killing was maintained at high potencyagainst the original stimulators (diamond and circle).

[0059]FIG. 10 is a graph depicting the relationship between cells perculture and the percent of non-responding cells of a CTL lineestablished from PBL of donor R (described in FIG. 1) by stimulationagainst third party EBV transformed stimulators (named S). Whenstimulating the cells of this CTL line against the host type PBL(patient No. 38), the specific killing was demonstrated to be negligible(circle) as is compared to that found against control cells (patient No.40, non-specific background) (square).

[0060]FIGS. 11a-b are bar graphs demonstrating the veto activity ofhuman anti-third party CTLs. FIG. 11a shows the inhibition of specificlysis at 1:25 and 1:50 veto to effector cell ratio, compared to acontrol experiment FIG. 11b) using the same veto cells in a non-relevantMLR, directed against an irrelevant stimulator bearings different HLAantigens.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] The present invention is of veto cell preparations, methods fortheir production and their use in transplantations, which veto cells canbe used to induce durable tolerance of donor organs, tissues or cellswithout inducing GVHD. Specifically, the present invention relates toveto cell preparations for use in transplantation, which veto cellpreparations include cells which are functional as veto cells but whichare substantially devoid of alloreactive cells and as such, whenintroduced into a recipient, these veto cells prevent graft rejectionwithout inducing GVHD.

[0062] The principles and operation of the present invention may bebetter understood with reference to the drawings and accompanyingdescriptions.

[0063] Before explaining at least one embodiment of the invention indetail, it is to be understood that the invention is not limited in itsapplication to the details set forth in the following description. Theinvention is capable of other embodiments or of being practiced orcarried out in various ways. Also, it is to be understood that thephraseology and terminology employed herein is for the purpose ofdescription and should not be regarded as limiting.

[0064] Early studies in murine models and more recent clinical data inheavily pre-treated Leukemia patients have shown that escalation ofhematopoietic progenitor cells can overcome major genetic barriers andenable rapid and durable engraftment of haploidentical 3-loci mismatchedtransplants without inducing GVHD. In vitro studies suggest that vetocells within the CD34 progenitors population likely mediate thisfacilitating effect.

[0065] The “megadose” concept was also shown recently to be useful fortolerance induction in sublethally irradiated mice by overcoming themarked resistance presented by the large number of lymphocytes survivingthe sublethal conditioning. Allogeneic chimera generated bytransplantation of a megadose of Sca1^(+ Lin) ⁻ cells, permanentlyaccept allogeneic donor type skin grafts.

[0066] However, the numbers required to attain this desirable goal maynot be easily collected from human donors.

[0067] Non-alloreactive, Peanut Agglutinin negative thymocytes from(host×donor) F1 which are non-alloreactive by virtue of their geneticcomposition, were also shown to enhance engraftment of T-cell depletedBM.

[0068] More recently these cells were shown to synergise with murineSca1⁺ Lin⁻ cells so as to reduce the minimal number of cells required toachieve induction of substantial mixed chimerism in the sub lethal mousemodel.

[0069] In humans, non-alloreactive T-cells can be generated by purginginterleukin-2 receptor (CD25) MLR reactive T-cells or by anergyinduction upon incubation with CTLA-4. However, these approaches, whichmake use of T-cell stimulation with the very antigens against whichtolerance is desired, might involve some risk for generating committedCTLs, if any of the CTL progenitors escape deletion or anergy induction.Once such anti-host CTLs are generated, it is very difficult to suppresstheir alloreactivity in vivo.

[0070] An alternative approach is based on earlier studies which showedthat CD8⁺ CTL clones possess extremely high veto activity.

[0071] Donor derived T-lymphocytes exposed to a third party antigenproved useful in generating CTLs effective in preventing or amelioratinggraft rejection, however, such CTL preparations included a substantialfraction of donor derived T-lymphocytes which could develop intoalloreactive donor CTLs, inducing GVHD.

[0072] Thus, all prior art approaches which utilize veto cells of T-cellorigin fail to provide adequate solution for inducing graft acceptancesince such veto cell preparations include a substantial amount ofalloreactive T-cells which induce GVHD in the host followingadministration.

[0073] While reducing the present invention to practice, donoranti-third party CTLs which are depleted of anti-host alloreactivitywere generated. In the preferred embodiments of the invention cellpreparations are also substantially depleted of CD4 T cells and/or CD56natural killer cells, preferably both.

[0074] As is further described in Examples 1 and 2 of the Examplessection below, depletion of cells having a potential of becomingalloreactive (anti-host) CTLs from a donor anti-third party CTLs cultureinvolved, in the specific example provided, initial IL-2 starvationwhich resulted in apoptosis of non-induced T-cells present in theculture. Studies utilizing such mouse and human CTL preparations whichwere performed in-vitro, demonstrated that such non-alloreactive CTLpreparations are depleted of cells having the potential of maturing intoanti-host CTLs.

[0075] Thus, according to one aspect of the present invention there isprovided a method of transplanting a transplant derived from a donorinto a recipient.

[0076] As used herein the terms “transplant” or “graft” refers to eitherallogeneic or xenogeneic transplants or grafts including, but notlimited to whole organs, such as for example, kidney, heart, liver orskin; tissues, such as, for example, tissues derived from an organ suchas a liver; or cells, such as, for example, immature hematopoicticcells.

[0077] As used herein the term “allogeneic” refers to as being from thesame species. As such an “allograft” is a transplant between twoindividuals of the same species which individuals display strong(unrelated individuals) or weak (haploidentical siblings)histocompatibility differences.

[0078] As used herein the terms “xenogeneic” or “heterogeneic” refer toas being from two different species. As such a “xenograft” or a“heterograft” is a transplant between two individuals of a differentspecies.

[0079] Thus, the method according to the present invention, which isfurther described and exemplified hereinbelow, can be utilized fortransplantation of an organ such as a kidney or a heart to a recipientsuffering from, for example, renal or heart failure, or for thetransplantation of liver, lung or skin tissue to a recipient sufferingfrom hepatic or lung failure or skin damage (e.g., burns).

[0080] The method described below can also be used, for example, fortreating a recipient suffering from a disease requiring immaturehematopoietic cell transplantation.

[0081] In the latter case, immature allogeneic or xenogeneichematopoietic cells (including stem cells) which can be derived, forexample, from bone marrow, mobilized peripheral blood (by for exampleleukapheresis), fetal liver, yolk sac and/or cord blood of the donor andwhich are preferably T-cell depleted CD34⁺ immature hematopoietic cells,can be transplanted to a recipient suffering from a malignant disease.Such a disease can be, but is not limited to, leukemia such as acutelymphoblastic leukemia (ALL), acute nonlymphoblastic leukemia (ANLL),acute myelocytic leukemia (AML) or chronic myelocytic leukemia (CML),and severe combined immunodeficiency syndromes (SCID), includingadenosine deaminase (ADA), osteopetrosis, aplastic anemia, Gaucher'sdisease, thalassemia and other congenital or genetically-determinedhematopoietic abnormalities.

[0082] Regardless of the transplant type, to avoid graft rejection, themethod according to the present invention also utilizes a novel vetocell preparation which includes non-alloreactive anti-third partycytotoxic T-lymphocytes (CTLs). Such CTLs are generated by conditioningdonor derived T-cells against third party antigens. This CTL cellpreparation functions in reducing graft rejection by the recipient.Further detail of such a CTL preparation is given in Examples 1-3 of theExamples section. Such preparations are depleted of T-lymphocytescapable of developing into alloreactive CTLs, thereby preventing orameliorating both graft rejection by the recipient and graft versus hostdisease.

[0083] As used herein the phrase “non-alloreactive” refers to havingsubstantially no reactivity against the donor or recipient.

[0084] According to the method of the present invention, these CTLs areadministered either concomitantly, prior to, or following thetransplantation of the donor transplant.

[0085] In the case of hematopoietic cell transplantation, the cell ratiobetween the cytotoxic T-lymphocytes and the immature hematopoietic cellsincluding stem cells is at least 1 to 100, preferably, in the range of 1to 1-5 to 1, more preferably about. Preferably, the cell number of thecytotoxic T-lymphocytes infused to a recipient is more than 5×10⁶/Kgbody weight and the immature hematopoietic cells including stem cells(CD34 cells) is more than 1.0×10⁶/Kg body weight.

[0086] As used herein the phrase “third party antigen or antigens”refers to antigens which are not present in either the donor orrecipient.

[0087] For example, third party antigens can be antigens of viruses,such as for example, Epstein-Barr virus (EBV) or cyto-megalo virus (CMV)or of third party cells (cells not from the donor or recipient). Viralantigens can be presented by cells (e.g., cell line) infected therewithor otherwise made to express viral proteins. Autologous antigenpresenting cells can be used to present short synthetic peptides fusedor loaded thereto. Such short peptides may be viral derived peptides orpeptides representing any other antigen. Dedicated software can be usedto analyze viral or other sequences to identify immunogenic shortpeptides, i.e., peptides presentable in context of class 1 MHC or class1 MHC.

[0088] Third party cells can be either allogeneic or xenogeneic withrespects to the recipient. Preferably, in the case of allogeneic thirdparty cells, such cells have HLA antigens different from that of thedonor but which are not cross reactive with the recipient HLA antigens,such that CTLs generated against such cells are not reactive againsttransplant or recipient antigens.

[0089] According to another preferred embodiment of the presentinvention the allogeneic third party cells are stimulatory cellsselected from the group consisting of cells purified from PBL, spleen orlymph nodes, cytokine-mobilized PBLs and in vitro expandedantigen-presenting dendritic cells (APC).

[0090] Third party antigens can be presented on the cellular, viral orbacterial surfaces or derived and/or purified therefrom. Additionally, aviral antigen can be displayed on an infected cell and a cellularantigen can be displayed on an artificial vehicles such as a liposome.

[0091] In addition, third party antigens can, for example, be proteinsextracted or purified from a variety of sources. An example of apurified protein which can serve as a third party antigen according tothe present invention is ovalbumin. Other examples are envisaged.

[0092] According to the present invention, the CTLs are preferablygenerated against third party cells or viruses or virally infected orviral peptides presenting cells and as such, the third party antigensutilized by the present invention are various native cellular or viralantigens or a combination of both which are displayed on the surface ofthe cell or virus.

[0093] Utilizing cells, viruses, virally infected or viral peptidespresenting cells as third party antigens is particularly advantageoussince such third party antigens include a diverse array of antigenicdeterminants and as such direct the formation of CTLs of a diversepopulation, which may further serve in faster reconstitution of T-cellsin cases where such reconstitution is required, e.g., following lethalor sublethal irradiation procedure. Furthermore, when CTLs are directedagainst viral infected cells, it is plausible to obtain at least somegraft versus cancer cells activity due to cross reactivity between viralantigens and cancer cell associated or specific antigens.

[0094] Thus, as already mentioned above, the method according to thisaspect of the present invention is effected by transplanting thetransplant into the recipient along with a non-alloreactive anti-thirdparty cytotoxic T-lymphocytes (CTLs) depleted of T-lymphocytes capableof developing into alloreactive CTLs, thereby preventing or amelioratingboth graft rejection by the recipient and graft versus host disease.

[0095] Since the non-alloreactive anti-third party CTLs are generated bydirecting T-lymphocytes derived from the donor against third partyantigen or antigens, the resultant CTLs prevent or ameliorate graftrejection by the recipient by trapping and killing recipient CTLprogenitors (responders) which are formed against the transplant.Furthermore, since the CTLs are generated against third party antigens,such CTLs are not active against recipient tissues or against the donortransplant. Since the anti-third party CTLs are depleted ofT-lymphocytes capable of developing into alloreactive CTLs, they preventor ameliorate graft versus host disease.

[0096] According to a preferred embodiment of the present invention themethod of transplanting a donor transplant further includes anadditional step in which the recipient is conditioned under sublethal,lethal or supralethal conditions prior to transplantation.

[0097] Such conditioning is dependent on the nature of the transplantand the condition of the recipient. The recipient may be conditionedunder sublethal, lethal or supralethal conditions, for example, by totalbody irradiation (TBI) and/or by treatment with myeloablative andimmunosuppressive agents according to standard protocols. For example, asublethal dose of irradiation is within the range of 1-7.5 Gy TBI, alethal dose is within the range of 7.5-9.5 Gy TBI and a supralethal doseis within the range of 9.5-16.5 Gy TBI. Examples of myeloablative agentsare busulphan, dimethyl mileran and thiotepa, and of immunosuppressiveagents are prednisone, methyl prednisolone, azathioprine, cyclosporine,cyclophosphamide, fludarabin, etc.

[0098] According to the method of the present invention, the recipientis preferably conditioned under sublethal conditions.

[0099] As already stated numerous times, the non-alloreactive anti-thirdparty CTLs according to the present invention are substantially depletedof T-lymphocytes capable of developing into alloreactive CTLs, and aretherefore said to be “non-alloreactive”. As such they differ and areadvantageous over prior art veto cells. Depletion of T-lymphocytescapable of developing into alloreactive CTLs is of particular advantagesince the presence of alloreactive cells in the recipient can lead tothe development of GVHD.

[0100] A non-alloreactive anti-third party CTLs cell preparation isgenerated as described above by directing donor derived T-lymphocytesagainst third party antigens and the preparation is depleted ofT-lymphocytes capable of developing into alloreactive CTLs.

[0101] According to one preferred embodiment of the present inventionthe depletion of T-lymphocytes capable of developing into alloreactiveCTLs is effected by depriving T-lymphocytes cultured in the presence ofthird party antigens of a factor which is (i) required for CTLsmaturation or protection from apoptosis; and (ii) secreted by maturingCTLs. Under such culturing conditions T-lymphocytes capable ofdeveloping into alloreactive CTLs undergo apoptosis, wherein maturingCTLs present in the culture survive factor deprivation since such cellsself secrete (autocrine) this factor.

[0102] The factor according to the teachings of the present inventioncan be for example, a cytokine, such as, but not limited to, IL2. IL2depravation of CTL preparations is described in detail in Examples 1-3of the Examples section below.

[0103] According to an embodiment of the present invention, depletion ofT-lymphocytes capable of developing into alloreactive CTLs is effectedby affinity labeling followed by label based separation. Thus, whenstimulated against host antigens ,a fluorescently labeled anti-CD69 oranti-CD25 antibody which specifically binds the unique activationantigen of T-lymphocytes and/or a fluorescently labeled anti-IL2 oranti-γINF which specifically binds cells secreting these cytokines, canbe used to separate anti-host T-lymphocytes from anti-third party CTLs,thereby deplete T-lymphocytes capable of developing into alloreactiveCTLs. Such specific labeling can be used to select anti-third party CTLprecursors prior to IL-2 starvation or as a substitute for IL-2starvation.

[0104] Such specific labeling can be used to select anti-third party CTLprecursors prior to IL-2 starvation or as a substitute for IL-2starvation.

[0105] According to still further features in the described preferredembodiments depletion of T-lymphocytes capable of developing intoalloreactive CTLs is effected by affinity purification.

[0106] For example, a substrate including an antibody or a ligandcapable of specifically binding a cell surface molecule displayed onlyby T-lymphocytes but not by CTLs or vice versa, can be used toeffectively deplete T-lymphocytes capable of developing intoalloreactive CTLs from the CTL preparation.

[0107] The affinity substrate according to the present invention can bea column matrix such as, for example agarose, cellulose and the like, orbeads such as, for example, magnetic beads onto which ananti-T-lymphocyte or an anti-CTLs antibody, as is described above, isimmobilized.

[0108] Thus, according to this aspect of the present invention,depletion of T-lymphocytes capable of developing into alloreactive CTLs,can be effected via column chromatography or magnetic bead separation.

[0109] The purpose in growing donor T cells sensitized against thirdparty antigen or antigens is to achieve a T cell preparation depleted ofalloreactive clones directed against host antigens. As is describedherein, the procedure includes two major steps. In the first, IL-2starvation is used to effect partial depletion of cells which cannotproduce their own IL-2, in the second major step anti-third party clonesare expanded vigorously with optimal doses of IL-2 so as to furtherdeplete remaining anti host clones. In is general, CD8 CTLs are directedagainst antigens presented in the context of HLA class I, while CD4helper T cells recognize antigens in the context of HLA Class II. Thelatter cells lack veto activity and therefore the procedure describedherein leads to minimal generation of such cells. However, it wasexperimentally found that even when using stimulation protocols favoringgeneration of CTLs against third party antigens, it is difficult toachieve high levels of CD8 CTLs at the end of the culture, unless CD4 Tcells and CD56 natural killer (NK) cells are removed 10-16 days afterthe IL2 starvation period (e.g., prior to stimulation with high levelsof IL-2). Thus in a preferred embodiment of the present invention CD8cells purification is effected by either negative selection for removingCD4 and CD56 cells or by positive selection with for retaining CD8cells. Such selection can be effected, for example, using affinity solidsupports including anti-CD8 antibodies (for positive selection) oranti-CD4 antibodies and anti CD56 antibodies (for positive selection).The solid support can be a column, beads or magnetic beads.Alternatively, such selection can be effected, for example, usingaffinity labeling followed by FACS separation employing fluorescentlylabeled anti-CD8 antibodies, anti-CD4 antibodies and/or anti CD56antibodies.

[0110] According to yet an additional aspect of the present inventionthere is provided a method of transplanting a transplant derived from adonor into a recipient. The method according to this aspect of thepresent invention is identical to the method described hereinabove withthe exception that the veto cells utilized are of non T-lymphocyteorigin.

[0111] A veto cell preparation according to this aspect of the presentinvention, includes donor derived, genetically modified non-T-cellsexpressing recombinant Fas ligand and recombinant CD8 antigen.

[0112] As is described in Example 2 of the Examples section, whilereducing the present invention to practice it was uncovered that CTLscan veto effectively CTL progenitors only if both Fas-L and CD8 areco-expressed on the former CTLs.

[0113] As such, donor derived non-T-lymphocyte circulatory cells, suchas, for example, monocytes, neutrophilles or basophilles, can also serveas effective veto cells provided these cells express and display CD8 andFas-L.

[0114] To express these proteins in such cells, the coding sequences ofFas-L and CD8 subunit o (Gene Bank accession Nos.: U11821 and M12825,respectively, both are incorporated herein by reference) includingappropriate leader sequences are isolated and each is ligated into asuitable expression cassette of an expression vector construct.

[0115] The vector constructs can then be co-introduced into the cell byany one of a variety of methods known in the art. Such methods can befound generally described in Sambrook et al., “Molecular Cloning: ALaboratory Manual”, Cold Springs Harbor Laboratory, New York, 1989,Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley andSons, Baltimore, Md., 1989, Chang et al., “Somatic Gene Therapy”, CRCPress, Ann Arbor, Mich., 1995, Vega et al., “Gene Targeting”, CRC Press,Ann Arbor Mich. (995), “Vectors: A Survey of Molecular Cloning Vectorsand Their Uses”, Butterworths, Boston Mass., 1988, and Gilboa et al.,Biotechniques 4 (6): 504-512, 1986, and include, for example, stable ortransient transfection, lipofection, electroporation, biolisticbombardment and infection with recombinant viral vectors.

[0116] Each nucleic acid construct according to this aspect of thepresent invention includes a promoter for regulate the expression ofFas-L or CD8. Such promoters are known to be cis-sequence elementsrequired for transcription as they serve to bind DNA dependent RNApolymerase which transcribes sequences present downstream thereof.

[0117] The promoter of choice that is used in conjunction with thisaspect of the invention can be any promoter suitable for expression inmammalian cells. Preferably the promoter utilized is a strong,constitutive promoter capable of expressing high levels of thetranscripts. In addition, to further increase transcriptional activitythe vector construct of the present invention may also include atranscriptional enhancer element.

[0118] The vector construct according to this aspect of the presentinvention preferably further includes an appropriate selectable marker.It will be appreciated that since two independent constructs areutilized herein, each construct includes a unique selectable marker,such that cell transformed with both constructs can be selected for.Alternatively, a single vector harboring both genes, each with its ownexpression regulatory sequences, is employed.

[0119] The vector construct according to the present inventionpreferably further includes an origin of replication in mammalian cellsand an appropriate selectable marker and origin of replication forpropagation in E. coli (i.e., shuttle vector). The vector construct ofthe present invention can be utilized for transient expression (i.e., nogenomic integration), or for integration into the genome of transformedcells and expression therefrom. The construct according to this aspectof the present invention can be, for example, a plasmid, a bacmid, aphagemid, a cosmid, a phage, a virus or an artificial chromosome.

[0120] It will be appreciated that CD8 and Fas-L can also be expressedfrom a single vector construct as a single chimeric transcript, providedthis transcript includes an IRES sequence for directing the translationof a second polypeptide encoded by the transcript.

[0121] Thus according to this aspect of the present invention the methodof transplanting a donor transplant utilizes a veto cell preparation ofgenetically modified non-T-cells expressing recombinant Fas ligand andrecombinant CD8 antigen.

[0122] It will be appreciated that such a veto cell preparation isinherently advantageous for inducing tolerance of transplants since sucha preparation does not include cells of T-lymphatic origin andtherefore, such veto cells inherently cannot induce GVHD.

[0123] In addition, since such cells are derived from the donor theyserve as excellent traps for CTL progenitors generated by the recipientas a response to the transplant and as such prevent or amelioratetransplant/graft rejection.

[0124] Thus, this aspect of the present invention describes analternative approaches to generating non-alloreactive anti-third partyveto cells. By expressing Fas-L and CD8, in a variety of cells, thesecells can serve as veto cells for antigens against which toleranceinduction is desired.

[0125] Similarly to the anti-third party CTLs described by other aspectsof the present invention, these ‘artificial’ veto cells could helpreduce the effective CD34 megadose requirements in mismatched leukemiapatients and may lead to safe mismatched hematopoietic transplants inpatients for whom the risk of supralethal radio-chemotherapy is notjustified, such as patients with thalassemia, sickle cell anemia andseveral enzyme deficiencies.

[0126] Furthermore, induction of substantial durable chimerism can beused, according to the present invention, to induce tolerance towardsorgan or tissue transplants, or as a prelude for adaptive cell therapyin cancer patients.

[0127] Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

[0128] Reference is now made to the following examples, which togetherwith the above descriptions, illustrate the invention in a non limitingfashion.

[0129] Generally, the nomenclature used herein and the laboratoryprocedures utilized in the present invention include molecular,biochemical, microbiological and recombinant DNA techniques. Suchtechniques are thoroughly explained in the literature. See, for example,“Molecular Cloning: A laboratory Manual” Sambrook et al., (1989);“Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M.,ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”,John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guideto Molecular Cloning”, John Wiley & Sons, New York (1988); Watson etal., “Recombinant DNA”, Scientific American Books, New York; Birren etal. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Example 1 Generation of Donor Anti-Third Party Mouse CTLs: IL-2Starvation Improves Deletion of Anti-Host CTL Precursors

[0130] Non-myeloablative conditioning protocols are currently exploredin elderly leukemia patients who have HLA identical donors. Engraftmentis mediated in these patients by the large number of T-cells present inthe unseparated peripheral blood progenitor preparations used fortransplantation. Thus, these transplants are associated with limitedGVHD and with anti-host reactivity leading to myeloablation of hosthematopoiesis. However, for patients who do not have a fully matcheddonor, T-cell toxicity is more pronounced and rather unacceptable.Removal of alloreactive T-cells from the transplant inoculum is likelyto weaken the engraftment potency and to result in a high rate of graftrejection. As such, and as described in this Example, anti-third partyCTL preparations devoid of cells capable of maturing into anti-host CTLswere established in order to reduce the rate of rejection and to preventinduction of GVHD.

[0131] While reducing the present invention to practice and as isspecifically shown herein, the inventors have uncovered that CTL vetocell preparations of a donor type, can be effectively depleted ofalloreactivity against host cells by stimulation against third partystimulators by initial exogenous IL-2 starvation. This depletion is dueto the fact that only activated CTL precursors (CT's) are capable ofsurviving the IL-2 starvation in the primary culture. Evidently, thisCTL preparation depleted of alloreactivity against the host couldpotentially facilitate the induction of donor type chimerism withoutGVHD in sub-lethally irradiated recipients.

[0132] In addition, this study, examines the mechanisms responsible forthe veto activity mediated by non-alloreactive anti-third party CTLs.

[0133] Materials and Methods:

[0134] Animals: 6-12 week old female mice were used. C3/HeJ, BALB/c,C57BL/6, gld/C3H/HeJ, lpr/C3H/HeJ were obtained from the Roscoe B.Jackson Memorial Laboratory (Bar Harbor, Me.). A breeding pair ofTransgenic H-2^(b) mice expressing the TCR-ab from the CTL clone 2C withspecificity for L^(d), was kindly provided by Janko Nikolic-Zugic,(Sloan-Kettering, NY). Progeny from these Tg mice and PO mice were bredat the Weizmann Institute Animal Breeding Center (Rehovot, Israel). Allmice were kept in small cages (five animals in each cage) and fedsterile food and acid water containing cyprofloxacin (20 μg/ml).

[0135] Preparation of non-alloreactive donor anti-third party CTLs:Splenocytes of Balb/c mice (donor responders) were harvested and singlecell suspensions were prepared. The cell suspensions were treated withTris-buffered ammonium chloride to remove red cells and the isolatedmononuclear cells (2×10⁶/ml) were stimulated with irradiated (20 Gy)C57BL/6 or C3H/HeJ (third party stimulators) splenocytes (2×10⁶/ml), andtreated with Tris-buffered ammonium chloride. Responders and stimulatorswere incubated in a complete tissue culture medium (CTCM) at 37° C. in a5% CO₂/air Heraeus incubator. 5 days following seeding, 20 U/ml of humanrhIL-2 (Eurocetus, Milan, Italy) were added to the mixed lymphocytereaction culture every 24 hours, 2 days later the medium was replaced bya fresh culture medium. 10 days following seeding, the MLR cultures wereharvested, fractionated on Ficol-Paque plus (Amersham Pharmacia BiotechAB, Sweden), analyzed by FACS for their CD8 level and added to the MLRcultures at different cell ratios, as described hereinbelow in theresults section.

[0136] MLR cultures and cytotoxicity assay: Spleen cells of C3H/Hej mice(responders) were harvested and single cell suspensions were prepared asdescribed above. The cells (2×10⁶/ml) were then stimulated withirradiated (20 Gy) Balb/c splenocytes (2×10⁶/ml), or with 2×10⁶/mlirradiated (20 Gy) SJL splenocytes. Four-six replicates per group werecultured in 96-well U bottomed plates in 0.2 ml CTCM, for 6 days at 37°C. (5% CO₂ atmosphere). ConA blasts generated from SJL or Balb/c spleencells (2 days in the presence of 2 mg ConA/2×10⁶ cells/ml) were labeledwith ⁵¹Cr (NEN, Boston Mass.). Cell-mediated lysis assay was performedusing variable numbers of MLR effector cells and 5000 target cells in96-well V-bottom plates. ⁵¹Cr release was measured after a 4-hincubation at 37° C. Results are expressed as a specific lysiscalculated as follows: % specific lysis=100×((experimentalrelease−spontaneous release)/(maximum release−spontaneous release)). Thestandard deviation (SD) of replicate values were consistently less than10% of the mean.

[0137] Veto activity of non-alloreactive donor anti-third party CTLs: Todetermine whether mouse non-alloreactive donor anti-third party CTLspossess veto activity, spleen cells from C3H/HeJ mice (2×10⁶/ml) wereincubated for 6 days with irradiated (20 Gy) allogeneic spleen cells(2×10⁶/ml) from Balb/c (H-2 matched) or SJL (H-2 mismatch) mice.

[0138] Non-alloreactive donor anti-third party Balb/c CTLs were added tothe MLC at a 1:100, 1:50 and 1:10 veto: responder cell ratio. Thekilling activity of the responder CTL-p was determined by ⁵¹Cr-releaseassay.

[0139] Inhibition of the veto effect of CTLs by anti-CD8 mAb: Vetoanti-third party CTLs from Balb/c origin were added to the MLR describedabove, at a final concentration of 2%. Anti-CD8 monoclonal antibody(kindly provided by Uli Hamerling, Sloan-Kettering, NY) directed againstLy-2.2 antigen expressed selectively on the veto cells and not on theeffector cells, was added to the MLR at different concentrations asdescribed in results, and the inhibition of the veto effect wasmonitored.

[0140] Deletion of anti-H2^(d) T-cells by non-alloreactive anti-thirdparty CTLs: Spleen cells of 2C Transgenic H-2^(b) mice expressing theTCR-ab with specificity for L^(d) mice (kindly provided by JankoNikolie-Zugic, Sloan-Kettering, NY), were treated as described above.The cells (2×10⁶/ml) were then stimulated with irradiated (20 Gy) Balb/csplenocytes (2×10⁶/ml) in the presence of 2% or 20% veto anti-thirdparty CTLs originated from Balb/c or from SJL (background control)splenocytes. Cultures containing 20% or 2% veto CTLs were continued for48 hours or 5 days, respectively, in 6-well plates. The deletion oftransgenic T-cells was monitored by cytoflourometry, measuring the levelof 2C transgenic cells specifically stained by the 1B2 antibody directedagainst the clonotypic anti-H-2L^(d) TCR.

[0141] Cytoflowmetry: FACS analysis was performed using a modifiedBecton Dickinson FACScan. Fluorescence data were collected using3-decade logarithmic amplification on 25-50×10³ viable cells asdetermined by forward-light-scatter intensity. Cells were stained with aCD8a (Ly-2)FITC, CD3e-PE, CD95 (Fas)-FITC (Phamingen) CD4-Qantum Red(sigma), 1B2 biotinated (kindly provided by Janko Nikolic-Zugic,Sloan-Kettering, NY), R-PE streptavidin (Jackson Immuno Research Lab.Inc.) Vectors for gene transfer: PLNGFP-mock vector was constructed bysubcloning gene encoding enhanced green fluorescence protein (EGFP,Clontech) from pEGFP-N1 (Clontech) into EcoRI and HpaI sites of pLXSNprovided by A D Miller, St. Jude Children's Research Hospital, Memphis,Tenn.) PLNGFPFas-L was also a derivative of pLXSN and was constructed bysubcloning of a fragment containing EGFP gene, internal nbosome entrysite (IRES) and murine Fas-L cDNA into BamHI and XhoI sites of pLXSN(LTR-EGFP-IRS-mFas-L-SV40p-NeoR).

[0142] Packaging of refroviral vectors and virus supernatant production:Plasmid DNA was transfected into ecotropic murine retroviral vectorpackaging cell line GP-E86 and stably transfected cells were selectedwith G418. High titter packaging cell lines were isolated by screeningindividual colonies. Retrovirus supernatant was prepared according tostandard procedure.

[0143] Gene transfer into splenocytes of Fas-L mutated C3H-gld mice: 50u/ml of human rhIL-2 were added to a CTL culture generated from “gld”spleen cells. Two days following addition, the stimulated CTLs wereresuspended in RPMI-1640 containing 5 mg/ml protamine sulfate and 50u/ml rhIL-2 and were incubated with viral supernatant which was addeddaily for 3 days. Forty eight hours following the last addition of theviral supernatant, the infected cells were selected in G418 (450 mg/ml)in the presence of rbIL-2 (50 u/ml) for one week and then tested fortheir veto activity in MLR as described above.

[0144] Experimental Results:

[0145] Veto activity of anti-third party CTLs: To evaluate the effect ofIL2 starvation on anti-host reactivity of non-alloreactive anti-thirdparty CTLs, Balb/c (H-2^(d)) splenocytes were stimulated, in thepresence or absence of recombinant human IL-2 (rhIL-2), againstirradiated (20 Gy) C57BL/6 (H-2^(b), third party) splenocytes in MLRculture for 4 days. On the second day or fifth day of the cell culturerhIL-2 was added and the cells were kept for additional six days. Afterculture, these cells were harvested and tested for their CTL-p frequencyagainst the intended host (C3H) or the third party used for stimulation(C57BL/6). As can be seen in FIG. 1, following IL2 starvation for 4 daysno anti-host CTLp could be detected while a significant frequency ofanti-host CTLp was documented when IL2 was added to the MLR culture onthe second day.

[0146] To evaluate the veto activity of such non-alloreactive C, Balb/cH-2^(d) splenocytes were stimulated against irradiated (20 Gy) C57BL/6H-2^(b) (third party) splenocytes in MLR culture for 10 days, addingrbIL-2 only on the fifth day of the cell culture. Following culturing,these cells were harvested and tested for their veto activity in MLRcultures of C3H/Hej H-2^(k) responder cells (host type) stimulatedagainst irradiated splenocytes from Balb/c (donor type), or in an SJLculture (non-relevant control, H-2^(s) type). The inhibition of killingactivity exerted by the veto cells on the responder cells was determined6 days post culturing by the ⁵¹Cr release assay.

[0147] As can be seen in FIG. 2a (17 different experiments) and in FIG.3 (one representative experiment), an addition of cells generated in aBalb/c CTL line directed against third party cells and cultured in theabsence of IL-2, inhibits the killing activity of the responder cells(C3H/Hej) which were stimulated against Balb/c splenocytes. In contrast,the addition of cells from the same CTL line to responder cells(C3H/Hej) that were stimulated against SJL splenocytes, did not affectthe responders killing activity. The specificity revealed by theseresults suggests that the non-alloreactive CTLs directed against thirdparty, indeed possess veto activity.

[0148] The dose response curve shown in FIG. 2b suggests that theanti-third party CTLs possess a very marked veto activity, attainingcomplete inhibition at a 1:50 veto/responder cell ratio. Addition ofveto cells at 5 fold higher concentrations leads to non-specificelimination of CTL-p's directed at targets not sharing the donor typeH-2 determinants (data not shown).

[0149] Optimal specific inhibition is achieved upon addition ofanti-third party CTLs, (at a 1:50 veto/responder cell ratio), betweenday 0 and day 2. Thereafter, the veto effect is markedly reduced (FIG.3). Thus, as previously suggested, veto cells probably inhibit or deletethe responder cells at the precursor level². Once these CTL precursorsdevelop into differentiated CTLs the veto cells can no longer exerttheir effect.

[0150] In addition, by using monoclonal antibodies directed against theCD8a Ly-2.2 allele expressed on the anti-third party CTLs but not on theresponder cells, confirmed previous observations¹⁸ that the CD8molecules of the veto cells participate in the deletion of the respondercells (FIG. 4).

[0151] The role of Fas-Fas-L apoptosis: The role of apoptosis inresponder cell deletion was confirmed by two approaches: (i) theaddition of the apoptosis inhibitor (BD-FmK) to the MLR culture led to acomplete inhibition of the veto effect (data not shown); and (ii) theaddition of anti-third party CTLs of Balb/c (H-2^(d)) origin, to an MLRculture of TCR transgenic C57B1/6 responders bearing the 2C TCR directedagainst H-2^(d), led to a marked and specific deletion of the transgenicresponders upon stimulation against Balb/c stimulators (FIGS. 5a-c).

[0152] Considering that two distinct major apoptosis mechanisms, namelyperforin mediated or Fas-Fas-L mediated apoptosis have been previouslydescribed, two different non-alloreactive anti-third party CTL linesfrom splenocytes of Fas-L deficient strain (C3H-gld, H-2^(k)) or fromsplenocytes of perforin deficient mice (PO, H-2^(bd)) were prepared.C3H-gld or PO-splenocytes were stimulated against irradiated C57BL/6H-2^(b) or DBA/1H-2^(q) splenocytes, respectively, as described for wildtype splenocytes (culturing for 10 days, without the addition ofexogenous rhIL-2 during the first 4 days). The use of veto cells of gld-C3H/Hej (H-2^(k) origin) and of PO (H-2^(bd) origin) necessitatedchanges in the design of the MLR responders and stimulators utilized.Thus, the gld mutant anti-third party CTLs were added to bulk MLRcultures in which Balb/c responders were stimulated against C3H/Hejsplenocytes. The PO knockout anti-third party CTLs were added to bulkMLR cultures in which C3H/Hej responders were stimulated against PO orSJL splenocytes.

[0153] As can be seen in FIG. 6a, the anti-third party CTLs originatingfrom C3H-gld lacked veto activity, whereas the CTLs originating fromperforin deficient mice did exhibit significant veto activity.

[0154] Additional evidence to the role of Fas-L in the veto effect wasafforded by gene transfer experiments in which gld-anti-third party CTLswere transfected with Fas-L, using a retroviral vector. As can be seenin FIG. 6b such transfected CTLs exhibited marked veto activity comparedto mock-infected anti-third party CTLs.

[0155] Using a similar approach, the role of Fas in the veto activityinduced by anti-third party CTLs was investigated. As can be seen inFIG. 7a, when Fas deficient C3H-lpr splenocytes were stimulated againstBalb/c in the primary MLR culture, the addition of Balb/c anti-thirdparty CTLs failed to inhibit the primary CTLs. In contrast, these cellsexhibited veto activity when added to primary MLR cultures of wild typeC3H responders. Similarly, a Fas antagonist (Fas fusion protein) alsoinhibited the veto effect of anti-third party CTLs (FIG. 7b).

[0156] FACS analysis of Fas during a typical MLR of 2C splenocytesagainst Balb/c stimulators (FIGS. 8a-d), revealed that Fas expression onCD8⁺ 1B2⁺ T-cells is markedly enhanced at 48 hr (FIG. 8b). Addition ofBalb anti-C3H (FIG. 8c), but not SJL anti-C3H CTLs (FIG. 8d), led to amarked deletion of the Fas⁺ activated CD8 T cells, illustrating thepotent veto activity and the remarkable specificity by which theanti-third party veto CTLs expressing Fas-L ²⁵, ²⁹ are capable ofselectively recognizing and killing Fas⁺ T-cells aimed against their H-2antigens.

[0157] Altogether these results suggest that the veto effect ofnon-alloreactive anti-third party CTLs is mediated by the simultaneousexpression of CD8 and Fas-L on the veto CTLs and the expression of Fason the effector CTL-p's directed against the antigens presented by theveto cells.

Example 2 Generation of Donor Anti-Third Party Human CTLs Depleted ofAnti-Host CTL Precursors

[0158] Following the demonstration described above that anti-third partyCTLs can be depleted of anti-host CTLp an attempt to apply this approachto human settings has been undertaken. It will be appreciated in thisrespect, and it is well accepted among art scholars, that it is not atall predictable that an immunogenic approach effective in mice wouldhave similar effectiveness in human beings.

[0159] Materials and Methods:

[0160] Establishment of anti-third party CTLs: Anti-third party CTLswere prepared by stimulating normal peripheral blood lymphocytes (PBL) sderived from a human donor against an Epstein-Barr virus (EBV)transformed cell line of known HLA type (hereinafter stimulator A). PBLswere cultured in growth complete medium+10% FCS (CM-RPMI 1640+2 mML-glutamine+100 U/ml penicillin+0.1 mg/ml streptomycin+2 mM HEPES+1 mMsodium pyruvate+0.1 mM non-essential amino acids+5×10⁻⁵ Mβ-mercaptoethanol) at 2×10⁶ cells/ml and were stimulated with 5×10⁴cells/ml irradiated (10,000 rad) stimulators.

[0161] Following 10 days of co-culture, the cells were harvested andlive cells were isolated on Ficoll-Paque gradients and re-stimulated,5×10⁶ cells/ml with 1.5×10⁵ cells/ml of stimulators. Four days later thecultures were treated with hrIL-2 at 20 U/ml, for the first time.

[0162] Thereafter, the cultures were treated three times per week, eachtime with 20 U/ml rhIL-2. The third treatment also included the additionof irradiated stimulators at a ratio of 4:1.

[0163] The CTL line, originally stimulated against stimulator S wastested for CTL-p responders against the original stimulator S or againststimulator No. 38 which represents the intended host.

[0164] The cells from this anti-third party CTL line were culturedeither with stimulator S or with stimulator 38, at 1×10⁶ cell/ml 1:1 for5 days. The cells were then harvested from the bulk culture and serialdilutions of CTL responder cells (4×10⁴ to 0.2×10³) per well, wereprepared with the original irradiated stimulators. Each well contains10⁵ of irradiated (30 Gy) stimulator 38 or 3×10⁴ (100 Gy) irradiatedstimulator S. The cultures were incubated for 7 days in completemedium+1%+20 U/ml rhIL-2.

[0165] Cytotoxicity was assayed with 5×10³ ⁵¹Cr labeled blasts (targetcells) from S, No. 38 and No. 40 (control for non-specific killing)donors.

[0166] Normal unseparated PBLs from the original donor of the cell linewere tested in parallel.

[0167] Experimental Results:

[0168] Non-alloreactive human CTL lines were established in-vitro bystimulating PBLs with EBV transformed cell line of known HLA type. Whilea significant level of anti-host CTLp was demonstrated in untreated PBLof donor A (FIG. 9), it was found that the anti-third party CTL linethat was generated from PBL of the same donor A was markedly depleted ofsuch cells (FIG. 10). These results indicate that the CTL preparation ofthis Example is depleted of T-cells having the potential of maturinginto anti-host CTLs (alloreactive CTLs) and is therefore advantageous asan agents for enhancing graft acceptance in humans.

CONCLUSIONS

[0169] Prior art approaches, which make use of T-cell stimulation withthe very antigens against which tolerance is desired, might involve somerisk for generating committed CTLs, if any of the CTL-p escape deletionor anergy induction. Once such anti-host CTLs are generated, it is verydifficult to suppress their alloreactivity in vivo.

[0170] A more recent approach, demonstrated that a “megadose” of cellscan be applied to induce tolerance in sublethally irradiated mice, andas such effectively overcome the marked resistance presented by thelarge number of lymphocytes surviving the sublethal conditioning.However, the number of cells required to attain this desirable goal maynot be easily collected from human donors.

[0171] Non-alloreactive anti-third party CTLs which can be used toenhance graft acceptance in mice were also shown, however, it was soonrealized that such CTL preparations are not depleted of T-cells capableof developing post transplantation into anti-host CTLs inflicting GVHD.

[0172] The present research demonstrates that donor anti-third partyCTLs can be depleted of anti-host alloreactivity if exogenous IL-2 isnot provided during the initial days of the bulk culture with thestimulator cells. Such non-alloreactive CTLs can enhance engraftment,without GVHD, of, for example, T-cell depleted BM originating from thesame donor.

[0173] The present study further uncovers the mechanism responsible forthe marked potent veto activity of such anti-third party CTLs.

[0174] By utilizing anti-CD8 antibodies, the present study demonstratesthat the expression of CD8 on the CTLs is crucial for CTL-p recognition.

[0175] By using anti-third party non-alloreactive CTLs generated fromspleen cells of different mutant mice the present study demonstratesthat the veto activity of these cells is mediated by apoptosis via theFas-Fas-L system and not by the perforin mechanism.

[0176] Thus, the results of the present study suggest that simultaneousexpression of both CD8 and Fas-L on the CTL surface and the expressionof Fas on the effector CTL-p is a prerequisite for the veto activity.

Example 3 The Primate Model

[0177] Materials and Methods:

[0178] Monkeys: Female cynomolgus monkeys (aged 1.5-2 years) weighing1.5-2.5 kg, are used as bone marrow recipients and MLC mismatched malecynomolgus monkeys weighing 6-9 kg are used as donors.

[0179] Conditioning regimen: A sublethal irradiation-based protocolutilized is as follows: Fludarabine (FLU) 40 mg/m² from day-9 (prior totransplantation) to day-5 (total amount 200 mg/m²); Rabbitanti-thymocyte globulin (ATG) 5 mg/kg from day-7 to -3 and 6.5 or 7 GyTBI (dose rate 0.15 Gy/min) delivered on day-1.

[0180] Peripheral blood mononuclear cells collection: RhG-CSF (20mg/kg/day) is administered to donor monkeys over a period of five daysin two daily subcutaneous injections. Two leukapheresis procedures areperformed between days 4 and 5.

[0181] Donors and leukapheresis: Healthy male cynomologus donors aresubjected to a double or triple set of 100-minute leukapheresis on aCobe Spectra cell separator, as described by Hillyer et al. [referenceNo. 27] with the following minor modifications: acid, citrate, dextrose(ACD)-to-blood ratio is 12:14 and centrifuge speed is 1,600 rpm.

[0182] Peripheral blood mononuclear cell processing: Peripheral bloodprogenitor cells (PBPC) preparations are depleted of T-lymphocytes usingthe E-rosetting technique, as previously described²⁶. Stem cells arethen purified by positive selection by using an avidin-biotinimmunoadsorbtion column (CEPRATE SC System, CellPro, Bothell, Wash.),according to the manufacturer's instructions. The number of CD34⁺ cellsis measured both in whole blood and in the leukapheresis product by flowcytometry. The T-lymphocytes before and after T-cell-depletion are alsomeasured, by using a phycoerythrin-coupled anti-CD2 monoclonal antibody.Aliquots are taken for differential cell counts, mAb staining and GFU-GMassay at each stage of processing.

[0183] Supportive care: Monkeys are cared for in laminar air-flow cagesuntil the neutrophils count recovered to 1×10⁹/L. All monkeys receivecyproxin and amphotericin B as gut preparation, combined antibiotics asprophylaxis against bacterial infection since the day of transplant, andfluconazole as antifingal prophylaxis. Acyclovir is administered forpreventing viral infection. Whole blood or component blood products aretransfused according to monkey's hematocrit and platelet count. Allblood products are irradiated with 25-30 Gy and filtered beforetransfusion.

[0184] Engraftment and chimerism deteriniation: Time to engraftment isassessed by determining the day after transplant on which monkeysdisplayed a level of 0.5 neutrophils×10⁹/L and 25×10⁹ platelets/Lindependent of transfusion support. Chimerism is assessed byquantitative polymerase chain reaction (PCR). Briefly, the PCR productsare subjected to 3% MetaPhor agarose (FMC) gel. The density of each bandfor Y specific DNA and competitor DNA is measured by using computerizeddensitometer and the target (Y specific)/competitor DNA (T/C) ratio foreach sample is calculated. At the point where Y specific and competitorDNA are in equivalence (i.e., ratio=1.0), the starting amount of Yspecific DNA prior to PCR is equal to the known starting amount ofcompetitor DNA.

[0185] Generation of donor-anti-third party CTLs: A slightly modifiedprocedure is used for preparation of monkey donor anti-third party CTLs.Peripheral blood cells of donor and third party monkeys are fractionatedon Ficoll-Paque plus and the isolated mononuclear cells, 1×10⁶/ml ofresponder cells and 2×10⁶/ml irradiated (20 Gy) third party stimulators,are cultured for 10 days in CTCM medium. Four days hours after seeding,human rhIL-2 (20 U/ml, Eurocetus, Milan, Italy), is added every 24hours.

Example 4 Preparation of Human CD8+ CTLs is Enhanced by Removal of CD4+and CD56+ Cells

[0186] It was observed that the level of CD8+ cells in the CTL culturesprepared essentially as described above is extremely variable.Therefore, selection of CD8+ cells by removal (negative selection) ofCD4+ and CD56+ cells, using antibodies linked to magnetic beads. As canbe seen in FIGS. 11a-b the cells obtained by this procedure exhibitedvery potent veto activity at a ratio of 1:25 and 1:50 veto to effectorcell ratio.

[0187] Treatment of donor cells: Anti third party CTLs were prepared bystimulating donor peripheral blood lymphocytes against EBV transformedhuman derived cell line of a known HLA type (stimulators) in 24—wellplates. The peripheral blood lymphocytes were cultured in growthcomplete medium supplemented with 10% fetal calf serum (CM-RPMI 1640, 2mM L-glutamine, 100 Units/ml penicillin, 0.1 mg/ml streptomycin, 2 mMHepes, 1 mM sodium pyruvate, 0.1 mM non essential amino acids, 5×10⁻⁵ Mβ2-mercaptoethanol) at 1×10⁶ cells/ml and stimulated with 2.5×10⁴cells/ml irradiated (10,000 Rad) stimulators, in 250 ml flasks at avolume of 50 ml.

[0188] After 10 days of co-culture, the cells were harvested and livecells were isolated on ficoll gradient and re-stimulated, the cells wereresuspended to a concentration of 5×10⁵ cells/ml and were re-stimulatedwith 1.5×10⁵ stimulator cells/ml.

[0189] Four days later, the cells were negatively selected for CD4+ andCD56+ cells with MACS beads (Miltenyi Biotec). The unbound cells wereresuspended at 1×10⁶ cells/ml and re-stimulated with 0.33×10⁶ stimulatorcells/ml. At this stage (following two weeks in culture) the cells weretreated with interieukin-2 (IL-2, Proleukin) at 600 Units/ml.

[0190] Thereafter, the cultures were treated three times per week, eachtime with 600U/ml IL-2. The third time also included the addition ofirradiated stimulators at a ratio of 4:1. Please note that during thefirst two weeks of culture, no IL-2 was added to the cells.

Example 5 Experiments in Human-Case Report

[0191] Treatment of donor cells: Anti third party CTLs were prepared bystimulating donor peripheral blood lymphocytes against EBV transformedhuman derived cell line of a known HLA type (stimulators), as describedin Example 4 above. The CTLs obtained were infused to a patient asdescribed bellow in order to tolerize the immune system of the patient,so as to facilitate engraftment of stem cells thereto.

[0192] The patient was diagnosed on May 1998 with mantle cell lymphomaThe patient was treated with 4 courses of PROMACE resulting in partialremission. During December 1998 the patient was treated with a high doseof Cytoxan (CTX) and autologous peripheral blood stem cells wereharvested and cryopreserved. During March 1999 the patient received anautologous transplant after conditioning with TBI-and thiotepa.Thereafter, complete remission has been achieved.

[0193] During October 1999 the patient relapsed with lympho-plasmacytoidinfiltration of the bone marrow. IgG/k serum levels were treated byplasmapheresis and dexametasone until July 2000.

[0194] During October 2000 the patient received an allogeneic mega dosestem cell transplant from a mis-matched haploidentical child. Thepatient was coditioned with melphalan 70 mg/sqm on day −12, thiotepa 7mg/kg body weight on day −11, fludarabin 40 mg/sqm from day −9 to day−6, Fresenius ATG 2.5 mg/kg body weight from day −9 to day −6. On daythe anti-third party CTLs were infused at a cell dose of 6.1×10⁶ CTL/Kgrecipient body weight. On day 0 the donor stem cells were infused at adose of 9.7×10⁶ CD34+ cells per Kg body weight (processed by CliniMACSand including 40,000 CD3T cells/Kg body weight). Conditioning as well asCTL infusion was well tolerated despite the pre-transplantcardio-pulnonary compromission of this old patient.

[0195] Neutrophil engraftment (500/mm³) was first found on day +8,980/mm³ on day +11 , 2,760/mm³ on day +14, and 5,370/mm³ on day +21.

[0196] Platelet reconstitution (35,000/mm³) was first achieved on day+18.

[0197] CMV antigenemia became positive (only 4 cells) on day +40(Patient was not under prophylactic treatment).

[0198] Gammapathy on electrophoresis, typical of Lymphoma patients, wasreduced to normal levels (before transplant—33.8%, 21% on day +20 posttransplant).

[0199] IgG level was reduced to normal levels (before transplant—2330mg/dl, 1550 mg/dl post transplant).

[0200] FISH analysis on day +20 post transplant revealed that all thecells in the peripheral blood and in the bone marrow were of donororigin.

[0201] Although the invention has been described in conjunction withspecific embodiments thereof, it is evident that many alternatives,modifications and variations will be apparent to those skilled in theart. Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications cited herein are incorporatedby reference in their entirety.

REFERENCES

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[0205] 4. Kaufman C L, Colson Y L, Wren S M, Watkins S. Simmons R L,Ildstad S T: Phenotypic characterization of a novel bone marrow-derivedcell that facilitates engraftment of allogeneic bone marrow stem cells.Blood 84:2436, 1994.

[0206] 5. Pierce G E, Watts L M: Do donor cells function as veto cellsin the induction and maintenance of tolerance across an MHC disparity inmixed lymphoid radiation chimeras? Transplantation 55:882, 1993

[0207] 6. Cobbold S P, Martin G, Qin S, Waldmann H: Monoclonalantibodies to promote marrow engraftment and tissue graft tolerance.Nature 323:164 1986.

[0208] 7. Kikuya S, Inaba M, Ogata H, Yasumizu R, Inaba K: Wheat germagglutinin-positive cells in a stem cell-enriched fraction of mouse bonemarrow have potent natural suppressor activity. Proc Natl Acad Sci USA85:4824, 1988.

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[0210]9. Pierce G E, Watts L M: Thy 1+ donor cells function as vetocells in the maintenance of tolerance across a major histocompatibilitycomplex disparity in mixed-lymphoid radiation chimeras. Transplant Proc25:331, 1993.

[0211] 10. Strober S, Palathumpat V, Schwadron R, Hertel-Wulff B: Clonednatural suppressor cells prevent lethal graft-vs-host disease. J Immunol138:699, 1987.

[0212] 11. Tschering T, Claesson M: Veto-like down regulation of Thelper cell reactivity in vivo by injection of semi-allogeneic spleencells. Immunology Letters 29:223, 1991.

[0213] 12. Cassell D J, Forman J: Regulation of the cytotoxic Tlymphocyte response against Qa-1 alloantigens. J Immunol 144:4075, 1990.

[0214] 13. Claesson M H, Ropke C: Antiself suppressive (veto) activityof responder cells in mixed lymphocyte cultures. Curr Top MicrobiolImmunol 126:213, 1986.

[0215] 14. Fink P J, Rammensee H G, Bevan M J: Cloned cytolyfic T cellscan suppress primary cytotoxic responses directed against them. JImmunol 133:1775, 1984

[0216] 15. Fink P J, Shimonkevitz R P, Bevan M J: Veto cells. Annu RevImmunol 6:115, 1988.

[0217] 16. Tscherning T, Claesson M H: Veto suppression: the peripheralway of T cell tolerization. Exp Clin Immunogenet 10:179, 1993.

[0218] 17. Claesson M H: Veto cell H-2 antigens: veto cell activity isrestricted by determinants encoded by K, D, and I MHC regions. CellImmunol 109:360, 1987.

[0219] 18. Sambhara S R, Miller R G: Programmed cell death of T cellssignaled by the T cell receptor and the alpha 3 domain of class I MHC.Science 252:1424, 1991.

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[0225] 24. Reisner Y, Li H W, Krauthgamer R, Marcus H, Bachar-Lustig E:Non-alloreactive donor anti-third party CTL's facilititate BM allograftacross major histicompatibility barriers in sublethally irradiated mice.Blood 92:265a, 1998.

[0226] 25. Reich-Zeliger S, Bachar-Lustig E, Zhao Y, Reisner Y:Enhancement of BM allografts by non-alloreactive donor CTL's: CD8binding and Fas-FasL apoptosis mediate the veto effect. blood 94:605a,1999.

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What is claimed is:
 1. A method of transplanting a transplant derivedfrom a donor into a recipient, the method comprising the steps of: (a)transplanting the transplant into the recipient; and (b) administeringto the recipient a dose including non-alloreactive anti-third partycytotoxic T-lymphocytes (CTLs), wherein said non-alloreactive anti-thirdparty CTLs are generated by directing T-lymphocytes of the donor againsta third party antigen or antigens, said dose being substantiallydepleted of T-lymphocytes capable of developing into alloreactive CTLs,thereby preventing or ameliorating both graft rejection by the recipientand graft versus host disease.
 2. The method of claim 1, wherein saiddose being substantially depleted of CD4 T cells.
 3. The method of claim1, wherein said dose being substantially depleted of CD56 natural killercells.
 4. The method of claim 1, wherein said dose being substantiallydepleted of CD56 natural killer cells and CD4 T cells.
 5. The method ofclaim 1, wherein depletion of T-lymphocytes capable of developing intoalloreactive CTLs is effected by deprivation of a factor which is (i)required for CTLs maturation; and (ii) secreted by maturing CTLs.
 6. Themethod of claim 5, wherein said factor is a cytokine.
 7. The method ofclaim 6, wherein said cytokine is IL2.
 8. The method of claim 7, whereinsaid dose being substantially depleted of CD4 T cells.
 9. The method ofclaim 7, wherein said dose being substantially depleted of CD56 naturalkiller cells.
 10. The method of claim 7, wherein said dose beingsubstantially depleted of CD56 natural killer cells and CD4 T cells. 11.The method of claim 1, wherein depletion of T-lymphocytes capable ofdeveloping into alloreactive CTLs is effected by affinity labelingfollowed by label based separation.
 12. The method of claim 1, whereindepletion of T-lymphocytes capable of developing into alloreactive CTLsis effected by affinity purification.
 13. The method of claim 1, whereinsaid donor is selected from the group consisting of an allogeneic donoreither HLA identical or HLA non-identical and a xenogeneic donor. 14.The method of claim 1, wherein the recipient is a human.
 15. The methodof claim 1, wherein said transplant is selected from the groupconsisting of cells, a tissue and an organ.
 16. The method of claim 1,further comprising the step of: (c) conditioning the recipient undersublethal, lethal or supralethal conditions.
 17. The method of claim 1,wherein the recipient and the donor are both humans.
 18. The method ofclaim 1, wherein said third party antigen or antigens is selected fromthe group consisting of third party cells, a cell antigen, a viralantigen, a bacterial antigen, a protein extract, a purified protein anda synthetic peptide presented by autologous or non-autologous presentingcells.
 19. The method of claim 18, wherein said viral antigen is an EBVor a CMV antigen.
 20. The method of claim 18, wherein said purifiedprotein is ovalbumin.
 21. The method of claim 18, wherein said thirdparty cells are allogeneic or xenogeneic cells with respect to therecipient.
 22. The method of claim 21, wherein said allogeneic cellshave HLA antigens different from that of the donor but which are notcross reactive with the recipient HLA antigens.
 23. The method of claim21, wherein said allogeneic cells are stimulatory cells selected fromthe group consisting of cells purified from peripheral bloodlymphocytes, spleen or lymph nodes, cytokine-mobilized PBLs and in vitroexpanded antigen-presenting dendritic cells (APC).
 24. The method ofclaim 1, wherein steps (a) and (b) are effected at the same time. 25.The method of claim 1, wherein steps (a) is effected prior to, orfollowing, step (b).
 26. A method of treating a recipient suffering froma disease requiring immature hematopoietic cell transplantation, themethod comprising the steps of: (a) conditioning the recipient undersublethal, lethal or supralethal conditions; (b) administering to therecipient a first dose including immature hematopoietic cells includingstem cells from an allogeneic or xenogeneic donor; and (c) administeringto the recipient a second dose including non-alloreactive anti-thirdparty cytotoxic T-lymphocytes (CTLs), wherein said CTLs are generated bydirecting T-lymphocytes derived from the donor against a third partyantigen or antigens, said second dose being substantially depleted ofT-lymphocytes capable of developing into alloreactive CTLs, therebypreventing or ameliorating both graft rejection and graft versus hostdisease.
 27. The method of claim 26, wherein said dose beingsubstantially depleted of CD4 T cells.
 28. The method of claim 26,wherein said dose being substantially depleted of CD56 natural killercells.
 29. The method of claim 26, wherein said dose being substantiallydepleted of CD56 natural killer cells and CD4 T cells.
 30. The method ofclaim 26, wherein depletion of T-lymphocytes capable of developing intoalloreactive CTLs is effected by deprivation of a factor which is (i)required for CTLs maturation; and (ii) secreted by maturing CTLs. 31.The method of claim 30, wherein said factor is a cytokine.
 32. Themethod of claim 31, wherein said cytokine is IL2.
 33. The method ofclaim 32, wherein said dose being substantially depleted of CD4 T cells.34. The method of claim 32, wherein said dose being substantiallydepleted of CD56 natural killer cells.
 35. The method of claim 32,wherein said dose being substantially depleted of CD56 natural killercells and CD4 T cells.
 36. The method of claim 26, wherein depletion ofT-lymphocytes capable of developing into alloreactive CTLs is effectedby affinity labeling followed by label based separation.
 37. The methodof claim 26, wherein depletion of T-lymphocytes capable of developinginto alloreactive CTLs is effected by affinity purification.
 38. Themethod-of claim 26, wherein said donor is selected from the groupconsisting of an allogeneic donor either HLA identical or HLAnon-identical and a xenogeneic donor.
 39. The method of claim 26,wherein the recipient is a human.
 40. The method of claim 26, whereinthe recipient and the donor are both humans.
 41. The method of claim 26,wherein said third party antigen or antigens is selected from the groupconsisting of third party cells, a cell antigen, a viral antigen, abacterial antigen, a protein extract and a purified protein.
 42. Themethod of claim 41, wherein said viral antigen is an EBV or a CMVantigen.
 43. The method of claim 41, wherein said purified protein isovalbumin.
 44. The method of claim 41, wherein said third party cellsare allogeneic or xenogeneic cells with respect to the recipient. 45.The method of claim 44, wherein said allogeneic cells have HLA antigensdifferent from that of the donor but which are not cross reactive withthe recipient HLA antigens.
 46. The method of claim 44, wherein saidallogeneic cells are stimulatory cells selected from the groupconsisting of cells purified from peripheral blood lymphocytes, spleenor lymph nodes, cytokine-mobilized PBLs and in vitro expandedantigen-presenting dendritic cells (APC).
 47. The method of claim 26,wherein said immature hematopoietic cells including stem cells arederived from the bone marrow, mobilized peripheral blood, fetal liver,yolk sac and/or cord blood of the donor.
 48. The method of claim 47,wherein said mobilized peripheral blood cells are obtained byleukapheresis of peripheral blood of the donor after stimulation with asuitable cytokine.
 49. The method of claim 26, wherein said immaturehematopoietic cells are T-cell depleted hematopoietic progenitor cells.50. The method of claim 49, wherein said T-cell depleted hematopoieticcells are CD34+ immature progenitor hematopoietic cells.
 51. The methodof claim 26, wherein a cell ratio between said cytotoxic T-lymphocytesand said immature hematopoietic cells including stem cells is at least 1to
 100. 52. The method of claim 26, wherein steps (b) and (c) areeffected at the same time.
 53. The method of claim 26, wherein steps (b)is effected prior to, or following, step (c).
 54. A method of producingnon-alloreactive anti-third party cytotoxic T-lymphocytes (CTLs), themethod comprising the step of directing T-lymphocytes against a thirdparty antigen or antigens, and substantially depleting T-lymphocytescapable of developing into alloreactive CTLs.
 55. The method of claim54, further comprising the step of substantially depleting CD4 T cellsfrom said T-lymphocytes.
 56. The method of claim 54, further comprisingthe step of substantially depleting CD56 natural killer cells from saidT-lymphocytes.
 57. The method of claim 54, further comprising the stepof substantially depleting CD56 natural killer cells and CD4 T cellsfrom said T-lymphocytes.
 58. The method of claim 54, wherein depletionof T-lymphocytes capable of developing into alloreactive CTLs iseffected by deprivation of a factor which is (i) required for CTLsmaturation; and (ii) secreted by maturing CTLs.
 59. The method of claim58, wherein said factor is a cytokine.
 60. The method of claim 59,wherein said cytokine is IL2.
 61. The method of claim 60, furthercomprising the step of substantially depleting CD4 T cells from saidT-lymphocytes.
 62. The method of claim 60, further comprising the stepof substantially depleting CD56 natural killer cells from saidT-lymphocytes.
 63. The method of claim 60, further comprising the stepof substantially depleting CD56 natural killer cells and CD4 T cellsfrom said T-lymphocytes.
 64. The method of claim 54, wherein depletionof T-lymphocytes capable of developing into alloreactive CTLs iseffected by affinity labeling followed by label based separation. 65.The method of claim 54, wherein depletion of T-lymphocytes capable ofdeveloping into alloreactive CTLs is effected by affinity purification.66. The method of claim 54, wherein said third party antigen or antigensis selected from the group consisting of third party cells, a cellantigen, a viral antigen, a bacterial antigen, a protein extract and apurified protein.
 67. The method of claim 66, wherein said viral antigenis an EBV or a CMV antigen.
 68. The method of claim 66, wherein saidpurified protein is ovalbumin.
 69. The method of claim 66, wherein saidthird party cells are allogeneic or xenogeneic cells with respect to therecipient.
 70. The method of claim 69, wherein said allogeneic cellshave ILA antigens different from that of the donor but which are notcross reactive with the recipient HLA antigens.
 71. The method of claim69, wherein said allogeneic cells are stimulatory cells selected fromthe group consisting of cells purified from peripheral bloodlymphocytes, spleen or lymph nodes, cytokine-mobilized PBLs and in vitroexpanded antigen-presenting dendritic cells (APC).
 72. A cellpreparation for transplantation to a recipient, said cell preparationcomprising donor derived non-alloreactive anti-third party cytotoxicT-lymphocytes (CTLs) directed against a third party antigen or antigens,said cell being substantially depleted of T-lymphocytes capable ofdeveloping into alloreactive CTLs.
 73. The cell preparation of claim 72,substantially depleted of CD4 T cells.
 74. The cell preparation of claim72, substantially depleted of CD56 natural killer cells.
 75. The cellpreparation of claim 72, substantially depleted of CD56 natural killercells and CD4 T cells.
 76. The cell preparation of claim 72, whereinsaid third party antigen or antigens is selected from the groupconsisting of third party cells, a cell antigen, a viral antigen, abacterial antigen, a protein extract and a purified protein.
 77. Thecell preparation of claim 76, wherein said viral antigen is an EBV or aCMV antigen.
 78. The cell preparation of claim 76, wherein said purifiedprotein is ovalbumin.
 79. The cell preparation of claim 72, wherein saidthird party cells are allogeneic or xenogeneic cells with respect to therecipient.
 80. The cell preparation of claim 79, wherein said allogeneiccells have HLA antigens different from that of the donor but which arenot cross reactive with the recipient HLA antigens.
 81. The cellpreparation of claim 79, wherein said allogeneic cells are stimulatorycells selected from the group consisting of cells purified fromperipheral blood lymphocytes, spleen or lymph nodes, cytokine-mobilizedPBLs and in vitro expanded antigen-presenting dendritic cells (APC). 82.A cell preparation for transplantation to a recipient, said cellpreparation comprising: (a) donor derived immature hematopoictic cellsincluding stem cells; and (b) donor derived, non-alloreactive anti-thirdparty cytotoxic T-lymphocytes (CTLs) directed against a third partyantigen or antigens, said cell preparation being substantially depletedof T-lymphocytes capable of developing into alloreactive CTLs.
 83. Thecell preparation of claim 82, substantially depleted of CD4 T cells. 84.The cell preparation of claim 82, substantially depleted of CD56 naturalkiller cells.
 85. The cell preparation of claim 82, substantiallydepleted of CD56 natural killer cells and CD4 T cells.
 86. The cellpreparation of claim 82, wherein said third party antigen or antigens isselected from the group consisting of third party cells, a cell antigen,a viral antigen, a bacterial antigen, a protein extract and a purifiedprotein.
 87. The cell preparation of claim 86, wherein said viralantigen is an EBV or a CMV antigen.
 88. The cell preparation of claim86, wherein said purified protein is ovalbumin.
 89. The cell preparationof claim 86, wherein said third party cells are allogeneic or xenogeneiccells with respect to the recipient.
 90. The cell preparation of claim89, wherein said allogeneic cells have HLA antigens different from thatof the donor but which are not cross reactive with the recipient HLAantigens.
 91. The cell preparation of claim 89, wherein said allogeneiccells are stimulatory cells selected from the group consisting of cellspurified from peripheral blood lymphocytes, spleen or lymph nodes,cytokine-mobilized PBLs and in vitro expanded antigen-presentingdendritic cells (APC).
 92. The cell preparation of claim 82, whereinsaid immature hematopoietic cells including stem cells are derived fromthe bone marrow, mobilized peripheral blood, fetal liver, yolk sacand/or cord blood of the donor.
 93. The cell preparation of claim 92,wherein said mobilized peripheral blood cells are obtained byleukapheresis of peripheral blood of the donor after stimulation with asuitable cytokine.
 94. The cell preparation of claim 82, wherein saidimmature hematopoietic cells are T-cell depleted hematopoieticprogenitor cells.
 95. The cell preparation of claim 94, wherein saidT-cell depleted hematopoietic cells are CD34+ immature progenitorhematopoictic cells.
 96. The cell preparation of claim 82, wherein acell ratio between said cytotoxic T-lymphocytes and said immaturehematopoietic cells including stem cells is at least 1 to 100.